| 其他摘要 | The successful cloning of mammals with somatic cell nuclear transfer (SCNT), especially the mice cloned from terminally differentiated lymphocytes and pos-mitotic olfactory neurons, provided unequivocal evidence for the nuclear totipotency of terminally differentiated cells. The SCNT technology has been successfully used in many species cloning, in production of transgenic, gene-knockout animal and model of human disease, and induced the breakthrough in therapeutic cloning and regenerative medicine with stem cells technology, thus holds great prospect for its application in future. However, many urgent problems , such as the low efficiency of success cloning, the developmental abnormalities in cloned embryos and offspring, and the losses throughout gestation, at birth and following birth, should be faced and resolved before the great promise become true. In mammalian preimplantation development, epigenetic reprogramming of DNA methylation, histone modifications and other epigenetic mechanism is essential to the establishment of development totipotency and pluripotency in normal embryo. More investigation on the reprogramming process will help to understand the reprogramming events occurred in cloned embryo; further provide insights into improving the reprogramming and development of cloned embryos. Rhesus monkey, an important kind of lab animal, is very significant for research of disease models and biological medicine. This study mainly focused on epigenetic reprogramming in cloned rhesus monkey embryo and treatment of somatic cells before nuclear transfer. The results were shown as follow: 1), the epigenetic reprogramming events, global methylation dynamics and histone H3 lysine 9 acetylation changes, were firstly described in nonhuman primate. Paternal genome was rapidly demethylated after fertilization and before DNA replication. Maternal genome was just gradually demethylated from 2-cell to early morula stage. Remethyaltion occurred in late morula and resulted into an asymmetric pattern of DNA methylation between hypermethylated trophectoderm and hypomethylated inner cell mass, which is contrary to the asymmetric pattern found in other mammals. 2), most cloned embryos were abnormal or delayed in epigenetic reprogramming. Many 2-cell (667%) and 8-cell cloned embryos showed higher methylation staining than their counterparts in IVF group. The methylation level of ICM nuclei in cloned blastocyst was always higher than IVF control, which might attribute to the development failure of cloned embryos after transfer into surrogate females. 3), In the experiment on treatment of donor cells before nuclear transfer, we found that several methods, including serum starvation and cycle inhibitors (DMSO, roscovitine, aphidicolin and indirubin), were all efficient in synchronizing cell cycle into G0+G1 stage. With BrdU labeling, their inhibitory effect on cell proliferation had been proved, moreover their effect was proved to be reversible. By TUNEL analysis, serum starvation induced more apoptosis in adherent cells (6%), whereas treatment with cycle inhibitors didn’t increase the occurrence of apoptosis compared to cycling cells control, which suggested these inhibitors treatment may be safe and efficient methods for synchronization of rhesus fibroblasts. |
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